The mechanisms regulating tumor susceptibility in cutaneous carcinogenesis reflect essential processes of the skin, and therefore investigation of immune contributions to these activities provides insight not only into the pathogenesis of skin cancer but also into basic cutaneous pathophysiology. Under two-stage chemical carcinogenesis with DMBA, a mutagenic polyaromatic hydrocarbon (PAH), followed by repeated applications of a pro-inflammatory tumor promoter, TPA, we observed that mice deficient in Langerhans cells (LC) are markedly resistant to tumor formation, and our findings have suggested an under- emphasized role of LC - their potential to enhance PAH-induced oncogenic H-Ras mutations. Recent work by others has revealed that the handling of PAHs by LC may have general implications for skin disease processes, including not only those relevant to chemically induced cancer, but also ultraviolet (UV)-induced p53 mutations. Thus, we propose to utilize the complimentary systems of chemical and UV- induced keratinocyte transformation, and novel animal models, to elucidate immune influences on cutaneous tumor initiation and promotion. Moreover, there is an irrefutable association of chronic inflammation and cancer in epithelial tissues continually exposed to irritants, toxins, and carcinogens. Thus, it is of major relevance that a further surprising finding of our studies of two-stage carcinogenesis has been the identification of a CD8+ T cell subset (T-pro) that promotes malignant progression. Thus, we propose to: (1) Investigate the role of LC in tumor initiation (mutagenesis) using the LC-deficient (Langerin-DTA) mouse, and study LC and other DC populations for their mutagenic capacity. For this, we designed a novel in vitro mutagenesis assay, and a novel real-time PCR method for the quantification of H-ras mutations. We will also use an inducible NKG2D-ligand mouse to investigate how major stimuli operative in the cutaneous environment may influence these activities so critical to tumor development. (2) Determine the contributions of LC to carcinogenesis during tumor promotion. For this we will use a new inducible LC- deficient (hLangerin-DTR) mouse to deplete LC after DMBA application, but before TPA promotion. (3) Determine the role of LC in UV-induced keratinocyte responses (apoptosis, p53 mutant clones, clonal expansion) critical to transformation. We will use the same constitutive and inducible models of LC deficiency as for chemical carcinogenesis. (4) Having recently delineated the expression pattern of CD8+ T-pro in association with malignant progression, we will further characterize their origin, differentiation, and potential for therapeutic manipulation. The elaboration of interactions between LC, keratinocytes, and T cells will substantially advance our understanding of carcinogenesis and basic skin biology; may provide insight into immune influences within other epithelia; and has implications for therapeutic regimens designed to modulate DC and/or T cell function. PUBLIC HEALTH RELEVANCE: This grant is about the regulation of skin cancer by immune cells. This research will study how the immune system locally influences tumors induced by chemical exposure, ultraviolet radiation, and inflammation.